Plant Symposia

P-1 P-3 Remembering Winter: Vernalization as an Environmentally Induced Epi- Diverse Small RNA-directed Pathways in Plants. ZHIXIN XIE. Dept. of genetic Switch. RICHARD AMASINO. Department of Biochemistry, Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131. University of Wisconsin, Madison, WI 53706. Email: amasino@ Email: [email protected] biochem.wisc.edu Most eukaryotic organisms possess highly conserved RNA silencing ma- Certain plants, such as biennials or winter annuals, require relatively long chinery that is associated with the formation of 21- ϳ 24-nucleotide small periods of cold exposure during winter to initiate ¯owering the following RNAs from precursor RNA molecules containing double stranded struc- spring. Cold exposure renders the meristem of such cold-requiring species tures. These endogenous small RNAs, which include microRNAs (mi- competent to ¯ower, and this acquisition of competence is known as RNAs) and small interfering RNAs (siRNAs) play important roles in vernalization. A vernalization requirement ensures that ¯owering does not regulation of gene expression, maintenance of genome integrity, control occur prematurely before the onset of winter. A similar cold response is of heterochromatin formation, and antiviral defense. Formation or activity bud dormancy; in many species that grow in temperate climates, bud of small RNAs requires factors belonging to gene families that encode dormancy is not broken until a the plant has ``counted'' a suf®cient num- DICER [or DICER-LIKE (DCL)], ARGONAUTE proteins and, in the ber of days of cold to ensure that any subsequent warn weather actually case of some siRNAs, RNA-DEPENDENT RNA POLYMERASE (RDR) indicates that spring has arrived. Our studies of vernalization in Arabi- proteins. Interestingly, unlike many animals, plants encode multiple DCL dopsis have revealed that meristem competence is a function of the ex- and RDR proteins. Recent genetic studies in Arabidopsis revealed that pression level of certain MADS-box genes such as FLOWERING LO- plants have evolved multiple functionally specialized small RNA path- CUS C (FLC) that act as repressors of ¯owering. Exposure to prolonged ways that require distinct DCL and RDR factors. Therefore, plants pro- cold causes an epigenetic switch of these MADS box genes to an unex- vide a unique system to study the evolution, diversi®cation, and func- pressed state, thus rendering the shoot apical meristem competent to ¯ow- tional adaptation of small RNA pathways. Unique functions associated er. This epigenetic switch is caused by covalent modi®cations to histones with distinct DCL and RDR factors in diverse small RNA-directed pro- of the chromatin of the ¯owering repressors. cesses will be presented.

P-2 P-4 Role of miRNAs and siRNAs in Abiotic Stress Responses. JIAN-KANG High Throughput Gene Assembly and Expression Using Viral RNA Rep- ZHU. Institute for Integrative Genome Biology and Department of Bot- licons Delivered by Agrobacterium. Y. GLEBA, S. Marillonnet, and V. any and Plant Sciences, 2150 Batchelor Hall, University of California, Klimyuk. Icon Genetics GmbH, Halle/Saale, D-06120, GERMANY. Riverside, CA 92521. Email: [email protected] Email: [email protected] Small non-coding RNAs ranging in size between 20 and 24 nucleotides Plant biotechnology relies on two processes for delivery and expression are important regulators of mRNA degradation, translational repression, of heterologous genes in plants, stable genetic transformation and tran- and chromatin modi®cation. These small RNAs can be broadly classi®ed sient expression with viral vectors or with Agrobacterium, but only the as miRNAs (microRNAs) and siRNAs (short interfering RNAs) based on transient routes provide a speed and a throughput necessary for fast re- their biogenesis. We found that the expression of some miRNAs and search and development studies. We developed an ef®cient, versatile and siRNAs in Arabidopsis plants are regulated by abiotic stresses such as high-throughput vector engineering and expression system based on in drought, soil salinity and cold temperatures. Data on the functional anal- planta assembly of functional viral vectors from separate pro-vector mod- ysis of several miRNAs and siRNAs using mutants and transgenic plants ules. With this system, we use agrobacteria to deliver various DNA mod- will be presented to support the regulatory roles of small RNAs in plant ules that are assembled inside the plant cell with the help of a site-speci®c adaptation to abiotic stresses. recombinase, integrase. The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating fully functional viral RNA replicons. The proposed protocol allows, by simply treating a plant with a mixture of two or more agrobacteria carrying speci®c prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform various engineering steps normally required with alternative methods. The process described is very fast (3±6 days); it provides very high protein yield (up to 5 mg per g fresh leaf biomass); and it is based on over 250 prefabricated genetic modules that allow to express a speci®c protein by adding speci®c promoters, signal/transit peptides, puri®cation tags, protein fusions, etc., in multiparallel studies.

In Vitro Biology Meeting 10-A Abstracts Plant Symposia

P-5 P-7 Signaling Networks Controlling Disease Resistance Responses in Ara- In Planta Transcriptional and Functional Patterns of an Agriculturally Rele- bidopsis. J. GLAZEBROOK, Raka Mitra, and Lin Wang. Department of vant R Gene. JAMES M. BRADEEN, B. P. Millett, and D. S. Mollov. De- Plant Biology, University of Minnesota, Saint Paul, MN 55108. Email: partment of Plant Pathology, University of Minnesota, St. Paul, MN 55108. [email protected] Email: [email protected] Plants respond to pathogen attack by activation of a battery of defense Potato late blight disease, caused by Phytophthora infestans, is among the responses. Activation is controlled by a complex regulatory network. Sec- most costly crop diseases worldwide. The disease affects both foliage and tors of this network are commonly de®ned based on the identities of tubers. Breeding efforts to improve potato late blight resistance have led to needed small molecule signals, salicylic acid (SA), jasmonic acid and the hypothesis that foliar and tuber blight resistance is conditioned by different related compounds (JA), and ethylene (ET). Genetic dissection of this resistance (R) genes. Previous research also documents changes in foliar blight resistance throughout plant development, suggesting R gene regulation or signaling network in Arabidopsis has identi®ed major regulatory genes function is dependent on physiological stage. The cloning of the foliar blight involved in these three sectors. A complete understanding of defense gene RB provides tools to test and explore these phenomena. We have de- network topology and function requires genome-scale analysis. To this veloped a highly sensitive RT-PCR assay to examine transgene RB expression. end, we have studied genetic perturbations of the network by transcrip- RB, like many R genes, is one of a cluster of sequence similar but functionally tional pro®ling using whole-genome microarrays. Similarity relationships disparate gene copies. However, our assay differentiates not only between RB among the transcriptional pro®les obtained from key mutants were used paralogs and RB alleles, but even between the RB transgene and the allele to create a basic model of network topology. It is obvious that effective from which it was cloned. We have also optimized whole plant and whole defense requires many regulatory and effector genes which have not yet tuber assays to functionally test for late blight resistance. The potato-P. in- been identi®ed. Many of these genes are expected to show increased festans pathosystem is an ideal system in which to test tissue-speci®c tran- expression in response to pathogen attack. In an effort to identify them, scriptional and functional regulation of R genes, since P. infestans is a natural plants with mutations in pathogen-induced genes have been screened for pathogen of two distinct plant tissues, leaves and tubers. We compared trans- enhanced disease susceptiblity phenotypes. Several important genes have gene RB transcription in foliage and tubers with results of our functional been identi®ed, including a cytochrome P450 monooxygenase required assays. Although the RB transgene is expressed in all plant tissues, leaves of for synthesis of the antimicrobial compound camalexin. transgenic plants are late blight resistant while tubers of transgenic plants are late blight susceptible. In related studies, we are exploring transcriptional and functional regulation of transgene RB throughout plant development. We have compared pre-¯owering, ¯owering, and post-¯owering transgenics using our RT-PCR and foliar blight resistance assays. Our research provides insight into strategies for the integration of transgene RB into potato disease management schemes. Future experiments include exploration of RB protein levels and comparison the disease response transcriptomes in various plant tissues and throughout plant development.

P-6 P-8 Pro®les in Scourge: Gene Expression Analysis of a Crop Killer. H. COR- Use of a High-performance, Custom Microarray for Elucidation of Sig- BY KISTLER and Kye-Yong Seong. USDA-ARS, Cereal Disease Lab- naling Networks Controlling Plant Defense Responses. M. Sato, R. Mitra, oratory, University of Minnesota, St. Paul, MN 55108. Email: R. van Poecke, J. Glazebrook, and F. KATAGIRI. Department of Plant [email protected] Biology, University of Minnesota, Microbial and Plant Genomics Insti- tute, St. Paul, MN 55108. Email: [email protected] Filamentous fungi are the most common and devastating causal agents of plant disease. Among the diseases of crop plants most important world- In systems analysis of a biological system, it is crucial to quantitatively wide is Fusarium blight of wheat and barley caused by the fungus Fu- and economically obtain a broad spectrum of information that character- sarium graminearum. With the availability of genome sequences for sev- izes the state of the system in detail. mRNA expression pro®ling could eral ®lamentous fungi including F. graminearum, large-scale functional yield data with high information content for the cost. We developed a genomics programs and genome-wide expression analysis is now possi- highly accurate, small-scale, oligonucleotide-spotted microarray (``mini''- ble. From the Fusarium sequence we have developed the ®rst Affymetrix array) for the purpose of analyzing Arabidopsis responses to pathogen GeneChip microarray based on the entire genome of a ®lamentous fun- infection. For a broad spectrum, we selected 464 genes that represent gus. To understand the early infection cycle of the pathogen, we moni- diverse expression patterns captured through many Arabidopsis GeneChip tored the RNA expression pro®les in newly formed spores, in maturing experiments related to biotic interactions. We employed 107 normaliza- spores and during the early stages of spore germination. We also exam- tion genes with a wide range of expression levels for accurate array-to- ined fungal gene expression during infection time-courses in wheat and array normalization. A hybridization signal from each spot was calibrated barley. The ability to detect fungal genes in planta is surprisingly sen- by a hybridization signal from the calibration oligonucleotide that was sitive even without efforts to enrich for fungal transcripts. These studies included in each probe solution when the array was spotted. Each probe will help to accelerate understanding of pathogenÐhost interactions by was spotted by four pens out of total 16 pens. The patterns of four pens elucidating expression of pathogenicity determinants in the fungus and for different probes were made overlapping in a symmetric manner, so disease response pathways in the plant. that the pen effect was removed from the signals by ®tting to a linear model. We obtained the correlation between technical duplicates better than 0.98. We conclude that we can omit technical replicates for a miniarray measurement. When the expression ratios calculated from the miniarray data were compared with those calculated from the Affymetrix ATH1 GeneChip data, the correlation was 0.88. This high quantitativity was con®rmed by qRT-PCR. We are currently using the miniarray for the purpose of sensitive screening and detailed characterization of reverse genetic Arabidopsis lines and detection, classi®cation, and mapping of naturally occurring alleles in loci controlling defense responses.

In Vitro Biology Meeting 11-A Abstracts Plant Symposia

P-9 P-11 Testing Methods for DNA and Proteins in Transgenic Crops. RAYMOND A Global Perspective on the Economic Impact of Transgenic Crop Va- D. SHILLITO. Bayer CropScience, Research Triangle Park, NC 27709. rieties. GREG TRAXLER. Department of Agricultural Economics, Au- Email: [email protected] burn University, AL 36849. Email: [email protected] The Agricultural, Biotechnology, Grain and Food and Feed Industries test Commercial transgenic crop varieties have been available in the US and for the presence of transgenic material in plants, seeds, grain and food. other countries since 1996. Twenty-one coutnries grew genetically mod- Testing starts with con®rmation of the transgenic nature of calli, and i®ed (GM) crops in 2005, but the countries of North and South America regenerated plants, and follows the plant through the breeding process accounted for 94%, of world GM crop area. Diffusion has been concen- until it is commercial. In addition, tests are needed for ensuring purity of trated among crops and traits as well; four crops (soybean, maize, cotton commercial seed, and once the crop leaves the farm gate it may be seg- and canola) and two traits (herbicide tolerance and insect resistance) ac- regated for use in certain markets. Testing is performed in order to satisfy count for 99% of GM crop area. This presentation reviews the adoption the needs of those involved in trade of grain and foodstuffs to comply of GM varieties, and surveys studies that have measured the level and with regulatory and labeling requirements that are in force in many coun- distribution of economic bene®ts from GM crops. The economic bene®ts tries. Agricultural Biotechnology companies develop methods as part of of the diffusion of GM crop varieties have been widely shared among the product development cycle. These are improved as the product comes farmers, industry, and consumers even though delivery has been through to market. Methods are also developed by commercial and government the private sector. GM crops have had a favourable environmental impact testing laboratories. This presentation will brie¯y describe some of the by facilitating reduced pesticide use and adoption of conservation tillage. tests available for detecting and quantifying DNA and proteins in trans- Key institutional factors in¯uencing GM diffusion, and the potential for genic plants, seeds, grain and food. Different test methods have different developing countries to bene®t from GM technology are discussed. costs and are used to obtain different results. Spraying plants (if they are herbicide resistant) shows if they are sensitive to herbicides. Protein- based methods such as LFS and ELISA can be used in several ways including to measure the expression of the trait (such as the PAT protein). PCR and other DNA-based methods can be used for screening or identify the actual event concerned, and RT-QPCR can estimate the amount present. It is also important to consider the resources required when deciding on the test method to use, as well as whether it needs to be done in the laboratory or the ®eld, and whether the necessary technical skills are available.

P-10 P-12 Applications of Testing Methods in the Grain Industry. R. W. GIROUX. Current Status and Impact of Commercial Plant Tissue Culture. STEVEN Cargill Incorporated, Wayzata, MN, 55391. Email: randal࿞giroux@ MCCULLOCH. Mountain Shadow Nursery LLC, Olympia, WA 98513. cargill.com Email: [email protected] Several countries have adopted or are in the process of developing leg- The present day successes with micropropagation or tissue culture prop- islation related to the approval of genetically modi®ed grain and grain agation of woody plants are due in part to the early advancements made products and/or the mandatory labeling of foods containing these prod- in the 1960s and 1970s with orchid, tropical and herbaceous plant tissue ucts. Most countries that have adopted an approval process or mandatory culture. Since the early 1980s the scope and breadth of commercial labeling schemes have set tolerances for events or thresholds for the ad- woody micropropagation in the United States has continuously expanded. ventious presence of transgenic material in grain products or the ®nal This expansion has been tempered by the higher costs associated with foods based on a %GM content. Once such regulations are enacted, in- this technology. Commercial woody plant propagation is limited to pro- dustry and government require analytical methods to monitor supply duction of crops that require or bene®t from this propagation method. An chains, certify product compliance, and enable enforcement. To test for overview of the history, crops produced and the current and future op- the presence of transgenic events or to measure the %GM requires vali- portunities of commercial woody plant micropropagation will be dis- dated methods that are ®t for the purpose and are suited to the testing cussed. environment. To meet this need, the food and feed supply chain are adopt- ing different strategies to make %GM determinations, including protein and DNA-based testing (PCR). During this presentation, several of the approaches that have been implmented will be discussed. In addition, discussion of emerging information on the impacts of genetics, processing, and method performance on these measurements will be discussed.

In Vitro Biology Meeting 12-A Abstracts Plant Symposia

P-13 P-15 Status of Commercial Tropical Foliage Plant Micropropagation. GARY Manufactured SeedÐAn Ef®cient Method for Delivery of Somatic Em- HENNEN. Oglesby Plants International, 2664 SR 71N, Altha, FL 32421- bryos to Nurseries. W. C. CARLSON, Weyerhaeuser Technology Center, 2848. Email: [email protected] WTC1B10, PO Box 9777, Federal Way, WA 98063±9777. Email: bill. [email protected] As the world's appetite for tropical ornamental plants continue to grow, regional production systems are becoming increasingly larger, sophisti- Manufactured seed can provide a delivery mechanism for cost effective cated and complex. Micropropagation has been an integral part of tropical implementation of clonal forestry. Manufactured Seed technology is de- foliage production for decades and as the tropical ornamental industry signed to sow somatic embryos into bare root and container nurseries, grows so does the requirements for larger volumes of high quality plants. enabling seedling growing in conventional facilities. Seed design, auto- At the same time, large consumers of tropical ornamentals are constantly mation and seedling cost near that of current orchard seed are all critical demanding lower price points, pushing commercial propagators to look to implementing the technology widely in forestry. Weyerhaeuser is in for alternative production methods and technologies requiring signi®cant late stages of development for low-cost, automated manufactured seed capital investments. This presentation will look at the great success story production processes for its seed design. The seed design incorporates the micropropagation industry has enjoyed working with tropical orna- many analogs of natural seed, including biodegradable materials. Manu- mentals and some of the challenges facing the industry in the near future. facturing processes involve automating all stages of the process, from somatic embryogenesis through sowing in the nursery. Demonstration machinery for manufacturing and advanced automation for many of the steps in seed manufacturing are completed and operating.

P-14 P-16 Temporary Immersion Bioreactor: An Ef®cient Technology For Scaling- Therapeutic Protein Expression in the Plant-based LEX System. VIN- up Plant Production. M. ESCALONA1, J. GonzaÂlez-Olmedo1, I. Cejas 1, CENT P. WINGATE, Biolex, 158 Credle Street, Pittsboro, NC 27312. C. AragoÂn 1, I. Capote 1, R. RodrõÂguez2,M.J.CanÄal 2, J. Sandoval 3,S. Email: [email protected] Roels 4, P. Debergh 4. 1Laboratory for Plant Cell and Tissue Culture, Bioplant Centre, University of Ciego de Avila, CUBA; 2Dept. B.O.S, Lemna, or duckweed, is a small aquatic plant that can be quickly trans- 3 formed to produce recombinant proteins in a contained and controlled University of Oviedo, SPAIN; and CORBANA, COSTA RICA; and TM 4Dept. of Plant Production University Gent, BELGIUM. bioprocessing environment. The bene®ts of the LEX System include a high transformation ef®ciency, rapid clonal growth in a contained and Temporary immersion has been shown to reduce problems usually en- controlled environment with high expression using a simple and inex- countered in liquid culture. Based on this concept, a collective of re- pensive production format. To date, over 25 different human therapeutic searcher belong to BioplantCenter adapted a semi-automated system for proteins have been produced by the LEX SystemTM, including several large-scale propagation of plants. This bioreactor has been named as Twin hard-to-make proteins and 10 different monoclonal antibodies. Flasks system (BIT௡) and grouped into the systems with complete immersion by pneumatic driven transfer of liquid medium without medium replenishment. BIT௡ is relatively simple and easy to use. They enable contact between all parts of the explants and the liquid medium. The culture environment is renewal by forced ventilation during each immersion period. For special type of plants, a forced aeration in the culture

recipient can be used. The injection of CO2 permits to improve the pho- tomixotrophic culture. BIT௡ has been used for in vitro commercial propagation of a wide range crops: Ananas, Saccharum sp, Musa sp, Colo- casia sp, Araceaes, Eucaliptys sp, Rosaceae, Bromelias, Paeony. In order to establish a micropropagation procedure and increase the ef®cacy of BIT௡-technology, different parameters should be optimized. Among them, the immersion time, immersion frequency, the volume of nutrient medium, the volume of culture container, the duration of proliferation phase, the use of plant growth retardant, the number of cycle in BIT௡. Plants regenerated by BIT௡ have not showed somaclonal variation de- tected by molecular probes and evaluations in the ®eld. The simplicity and low cost of BIT௡ is compatible with large-scale propagation. It permits important lower labor, better biological yield and consecutively re- duces production cost.

In Vitro Biology Meeting 13-A Abstracts Plant Symposia

P-17 P-19 Production of Biodefense-Related Proteins in Tobacco. K. WYCOFF. An Overview of the Orchid-fungal Symbiosis in Nature, and its Appli- Planet Biotechnology. Email: [email protected] cation In Vitro to Promote Conservation. L. W. ZETTLER. Orchid Re- covery Program, Department of Biology, Illinois College, Jacksonville, Botulism and anthrax are lethal bacterial diseases whose symptoms are IL 62650. Email: [email protected] caused mainly by biological toxins. Botulinum neurotoxin and anthrax spores are considered potential bioweapons, and current defenses and In nature, the orchid life cycle is initiated and closely tied to the avail- treatments are inadequate to protect a large target population that is likely ability of fungi in various substrates (e.g., decaying wood, Sphagnum to include civilians. Both therapeutic and prophylactic treatment with moss). For reasons not yet understood, fungi infect orchid seeds forming monoclonal antibodies have been shown to be more effective and safer coils of hyphae (pelotons) within the embryo, protocorm, seedling, and than alternatives, but stockpiling suf®cient doses is likely to be expensive. mature plant. Once lysed, these structures serve as a critical energy (car- Plants as a production system offer the promise of lower costs. We have bohydrate) source until photosynthesis is initiated, and give these plants demonstrated that plant-made monoclonal antibodies to botulinum neu- an alternative nutritive strategy (ϭmycotrophy) into adulthood. In terres- rotoxin A are effective and safe in a mouse model. We have also produced trial orchids, mycotrophy is believed to supplement photosynthesis, but in plants a fusion protein based on a human anthrax toxin receptor and serves as the primary source of carbon for achlorophyllous species. Epi- shown it to be an effective decoy, blocking the toxic effect of anthrax phytic orchids, which may rely on mycotrophy to a lesser extent, utilize lethal toxin in vitro. fungi as a source of free water to resist desiccation on arboreal substrates. Given the importance of fungi in situ, the preservation of orchid seeds alone has raised conservation concerns, and has prompted interest in using fungi in vitro for propagation (ϭsymbiotic seed germination). This presentation will discuss ongoing global efforts to cultivate orchids threat- ened with extinction using fungi (e.g., Platanthera holochila, endemic to Hawaii), and will discuss the potential ecological consequences of this practice.

P-18 P-20 Transgenic Expression and Recovery of Biologically Active Recombinant Symbiotic and Asymbiotic Orchid Seed Germination as Tools in Con- Human Insulin from Arabidopsis thaliana Oilseeds. ELIZABETH W. servation. S. L. STEWART. Environmental Horticulture Department, Uni- MURRAY1, Cory L. Nykiforuk1, Joseph G. Boothe1, Richard G. Keon1, versity of Florida, Gainesville, FL 32611. Email: [email protected]¯.edu H. Joseph Goren2, Nancy A. Markley1, Maurice M. Moloney11Sem- BioSys Genetics Inc, 110, 2985-23 Ave NE, Calgary, Alberta. T1Y 7L3 The loss of orchid-rich habitat worldwide, as well as the restoration of many historical orchid habitats has demonstrated the need for researchers CANADA and 2Department of Biochemistry and Molecular Biology, University of Calgary, Faculty of Medicine, Calgary, Alberta T2N 4N1 to develop appropriate propagation methods for the Orchidaceae. Tradi- CANADA. Email: [email protected] tionally, seed germination has been seen as the most ef®cient and effective method of plant production for orchids-asymbiotic seed germination With the emergence of new delivery technologies and the rise in inci- historically being the most commonly used method for both conservation dence of diabetes, the demand for affordable insulin will soon exceed the or commercial ends. While asymbiotic germination does represent a sim- current manufacturing technologies. To address this potential production ple method for the mass production of orchids, it does not take into shortfall, we have developed a novel expression and puri®cation tech- account the physiological need for fungal mycorrhizae during seed ger- nology to produce human insulin in oilseed plants. Recombinant human mination and, possibly, during later life stages. Only symbiotic orchid precursor insulin was expressed in Arabidopsis oilseeds and was found seed germination accounts for this fungal partner in both germination and to accumulate at a level of 0.13% of the total seed protein. The precursor subsequent development of the orchid plant. Both the asymbiotic and protein was digested with trypsin, in vitro, to produce mature insulin with symbiotic methods represent possible avenues in producing orchid seed-

a mass identical to that of the predicted DesB30-insulin product. In ad- lings for use in conservation and restoration efforts. The bene®ts and dition, we con®rmed the biological activity of this plant produced insulin shortcomings of both methods will be discussed. using cell based assays of receptor phosphorylation and insulin tolerance tests in mice.

In Vitro Biology Meeting 14-A Abstracts Plant Symposia

P-21 P-23 In Vitro Strategies for Conservation of Madagascar's Endemic Orchids. MAR- From Crops to Biore®neries. O. V. SELIFONOVA. Biotechnology De- GARET MARY FROM. Omaha's Henry Doorly Zoo, Center for Conservation and velopment Center (BioTDC), Cargill Incorporated, Minneapolis, MN Research, 3701 S 10th Street, Omaha, NE, 68107. Email: [email protected] 55440. Email: olga࿞[email protected] Madagascar's orchid species represent some of the most critically endangered mem- Alternative or genetically modi®ed crops have become an integral part bers of the family on earth today. The high degree of endemism; estimated at more than 80%, the richness of diversity among Madagascar's orchids, and the continuing of modern biotechnology. Current methodology for introduction of for- threats to the country's remaining natural areas, make the country's conservation eign genes into economically important plant species can be used not action plan imperative if these species are to survive for the future. Ex situ con- only for crop improvement and production of novel products in plants, servation measures present an opportunity to help preserve the island's more than but also for alteration of biomass composition that is tailored for needs one thousand orchid species. Few orchids are being propagated in Madagascar, and of industrial bioprocessing. Biobased industrial chemicals derived from in vitro germination protocols were not previously worked out for the majority of biomass, vegetable oils and carbohydrates have all prerequisites to com- the native orchid species. Reintroduction of orchids to the country's remaining pete with the scale, ¯exibility and ef®ciency of the petrochemical indus- natural areas was virtually non-existent prior to this collaborative effort. Orchid try. 3-Hydroxypropionic acid (3-HP) is one of the examples of biobased populations are rapidly declining in many parts of the country. A partnership between Omaha's Henry Doorly Zoo in the USA; which provided all in vitro bio- industrial chemicals that can be produced from renewable sugars. The technology training, and the University of Antananarivo, Madagascar, successfully chemical is not commercially available in large quantities, but has the propagated many endemic Malagasy orchids at a great distance from the seed potential to be a new industrial platform chemical. 3-HP has two func- source. A strategy of in vitro culture that allows the orchids to be returned to tional groups and can be readily converted to a range of important chem- Madagascar while still in sterile cultures, minimizes the risk of introducing path- icals, such as acrylic acid and 1,3-propanediol. Currently, no known or- ogens to the natural environment. The micropropagated orchids are treated to lower ganism makes 3-HP as a metabolic end-product. Using various sources nutrient and sucrose levels in vitro prior to their return to protected areas, and are of genetic information we designed and constructed several different met- acclimatized to ex vitro conditions when they arrive in Madagascar. Timing the abolic routes to 3-HP. We then selected one route for further development reitroductions to coincide with the wet season helps assure a higher survival rate. based on a series of design criteria, including high theoretical yield from Additionally, cryopreservation protocols were developed at Omaha's Henry Doorly Zoo laboratory, which are preserving orchid seed germplasm for long-term con- sugars. This route also required signi®cant use of directed evolution to servation purposes. Species representing 14 seperate orchid genera have been suc- modify and improve enzyme activities. This work provides an example cessfully micropropagated with more than 600 individual orchids already returned of the use of modern biotechnology to develop sustainable routes to in- to Madagascar from the zoo, where they have been acclimatized and reintroduced dustrial chemicals from crop-based renewable resources. directly into Ranomafana National Park through cooperation with the government and local residents in Madagascar. In some cases no natural seedling recruitment has occurred near the mother plants ®rst observed in 2000, and the seedlings successfully reintroduced in 2004 and 2005 represent the only juvenile specimens for some of the populations being studied.

P-22 P-25 Expanding the Utility of Alfalfa. R. A. DIXON, F. Chen, Y. Pang, and Development of Roundup Ready௡Alfalfa Varieties. MARK MCCASLIN, Peter Reisen, G. Peel. Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Holly Deery, Sharie Fitzpatrick, and Stephen Temple. Forage Genetics International, P.O. Sam Noble Parkway, Ardmore, Oklahoma, 73401. Email: radixon@ Box 339, Nampa, ID 83653±0339. Email: [email protected] noble.org Roundup Ready௡ Alfalfa (RRA) was jointly developed by Forage Genetics International (FGI) and Monsanto Company. In 1998 FGI produced ϳ 150 transgenic alfalfa plants Alfalfa (Medicago sativa) is perhaps the world's major forage legume. expressing the CP4 EPSPS gene, driven by an enhanced version of the Figwort mosaic Forage quality itself is an important target for biotechnological improve- virus promoter. Alfalfa is a cross pollinated autotetraploid plant. To achieve high trait ment. Many years of research have identi®ed lignin as an impediment to purity (i.e. Ͼ90% Roundup tolerant plants in a RRA variety) a two transgenic event, dihomogenic breeding strategy was adopted. From the initial 150 transgenic plants two forage digestibility, and lack of condensed tannins as promoting pasture commercial transgenic events were selected based on tolerance to Roundup herbicide, bloat and limiting nitrogen nutrition. Recent progress on understanding agronomic performance, molecular characterization and reproductive stability of the and manipulating the pathways leading to lignin and condensed tannins transgene insert. Event-speci®c PCR markers were developed to genotype Roundup will facilitate engineering of alfalfa and other forage legumes for reduced Ready௡ plants carrying one or both transgenic events. Both Roundup Ready௡ Alfalfa bloating potential and improved digestibility and palatability. The above (RRA) events were introgressed into elite FGI breeding populations using a modi®ed traits involve natural products, which are made by plants for their own backcrossing breeding strategy (MBCx). Four to six cycles of modi®ed backcrossing were used to develop FD3 to FD9 type breeding populations. Advanced MBCx lines health care (ie to ward off pests and pathogens). However, the impact of containing the two commercial events (RRA event B and RRA event D) were kept plant natural products on human health is increasingly recognized. With separate in the ®eld and in the greenhouse. Elite parents were selected from breeding the advent of genetic and genomic approaches, the synthesis of many nurseries established with seedlings from these advanced MBCx lines. Crosses between plant natural products is now understood at a level to permit their engi- elite parents containing event B were crossed to elite parents containing event D. The neering in crop plants. Alfalfa has been engineered as a delivery vehicle 1,1 dihomogenic progeny, containing a single copy of each event, were identi®ed using event speci®c markers. Based on pedigree and agronomic characteristics sets of these for iso¯avone phytoestrogens and the antioxidant epicatechin. Many other dihomogenic plants became Syn0 parents for RRA experimental varieties. In 2003 Syn1 compounds of nutraceutical or pharmaceutical value could be pro®tably seed of several RRA experimental varieties (FD3-FD8) was produced in isolation near made in alfalfa. Forage crops with genetically improved quality (output) Nampa, ID. Trait purity of these populations ranged from 92.5 to 94.8%, with a mean traits will bene®t both the health of the animals that consume them and of 93.8%Ðvery near the theoretical, expected value of 93.7%. In August and September the environment through reductions in waste excretion and greenhouse 2003 multiple location variety trials were established to evaluate agronomic performance and crop safety of RRA experimental varieties. Forage yield and quality data collected gas emission. Furthermore, the same modi®cations to lignocellulose that in 2004 and 2005 showed performance of the RRA experimental varieties, under con- improve digestibility may also improve the processing ability of forage ventional herbicide treatment, equal to or better than the commercial check varieties. crops for biofuel production. Crop safety of the RRA experimental varieties sprayed with maximum labeled rate of Roundup herbicide was excellent. Following U.S. deregulation of the two transgenic events in June 2005, ®fteen RRA varieties were commercially released in August, 2005. * ௡Roundup and Roundup Ready are registered trademarks of Monsanto Technology LLC.

In Vitro Biology Meeting 15-A Abstracts Plant Symposia

P-26 P-28 Development and Characterization of Alfalfa Populations Tolerant to Gly- Wide-cross Whole-genome Radiation Hybrid Mapping in Cotton. DAVID phosate. GLEN ROGAN, Sharie Fitzpatrick, Todd Pester, Daniel Kendrick, M. STELLY. Dept. of Soil and Crop Sciences, Texas A&M University, Michael Horak, Melinda McCann, Karu Karunanandaa, Stephen Temple, and College Station, TX USA 7843-2474. [email protected] Mark McCaslin. Monsanto Company, 800 N Lindbergh Blvd, St. Louis MO 63167. Email: [email protected] The development of animal genomics was catalyzed strongly by the advent of whole-genome in vitro radiation hybrid (RH) mapping. In lieu of Monsanto Company and Forage Genetics International have developed vari- a comparable in vitro plant system, we opted to develop an in vivo sys- eties of Roundup Ready* alfalfa that are tolerant to glyphosate, the active ௡ tem, based on interspeci®c hybridization between Gossypium hirsutum L. ingredient in Roundup agricultural herbicides. Roundup Ready alfalfa was and G. barbadense L, using irradiated pollen. The underlying rationale developed using Agrobacterium-mediated plant transformation to stably in- was that [1] an egg cell nucleus could be used to ``rescue'' the irradiated corporate into the alfalfa genome a coding sequence that encodes for a glyphosate-tolerant form of the enzyme 5-enolpyruvylshikimate-3-phosphate syn- sperm nucleus, [2] the interspeci®c nature of hybridization could provide thase (EPSPS). Roundup Ready alfalfa varieties enable growers to apply allelic diversity, [3] the gamma irradiation could segment the inherited Roundup agricultural herbicides from planting through ®ve days before cut- paternal genome, and [4] the unique origin of each radiation hybrid would ting, providing an additional tool for improved weed control, excellent crop greatly reduce and potentially eliminate chimerism within individual pan- safety and preservation of yield potential and forage quality. Characterization el members. Initial experiments established a workable irradiation treat- of Roundup Ready alfalfa plants was performed as part of the food, feed and ment and culminated in the establishment of a 5-Krad wide-cross whole- environmental safety assessment performed prior to regulatory submissions. genome radiation hybrid (WWRH) panel of G. hirsutum. RH maps were Key questions addressed were whether the introduced trait or the transfor- derived by analysis of SSR data using RHMAP software, and were com- mation and regeneration process impacted the phenotype of alfalfa or compared to locally available linkage maps and hypoaneuploid cytogenetic position of forage and whether there were any signi®cant environmental im- stocks. While the 5-Krad (50-Gy) RH panel was capable of detecting pacts associated with the introduction of Roundup Ready alfalfa. Information synteny among unlinked primitive linkage maps, the map resolution and data on the introduced trait indicate that the CP4 EPSPS protein is safe seemed to be too low in some areas. We therefore re-investigated higher for consumption. Other than the introduction of tolerance to glyphosate, there dosages, and constructed and characterized a second WWRH panel after were no biologically meaningful phenotypic differences between Roundup 8-Krad segmentation of the G. barbadense L. genome. Limited numbers Ready alfalfa populations and the alfalfa control or conventional reference of strategically chosen markers were used to compare WWRH mapping varieties. The levels of key nutrients and components in Roundup ready alfalfa results to the 5-Krad WWRH maps and to linkage maps. The results forage were comparable to the control and within the population of commer- indicate WWRH mapping can contribute signi®cantly to cotton genomics. cially available alfalfa varieties. Collectively, these results establish that Roundup Ready alfalfa is safe for use as feed or food and for release into the environment. *௡Roundup and Roundup Ready are registered trademarks of Monsanto Technology LLC.

P-27 P-29 Impact of Yieldguard Rootworm on Corn Rootworm Control. S. C. JOHNSON, G. R. Evolution of Chromatin Structure and Function. S. M. Kaeppler. Depart- Heck, and T. T. Vaughn. Monsanto Company, Chester®eld, MO 63017. Email: ment of Agronomy, University of Wisconsin, Madison, WI 53706. Email: [email protected] [email protected] In 2003, the Monsanto Company commercialized YieldGuard௡ Rootworm (YGRW) protected corn hybrids. These hybrids use a variant Bt Cry3Bb1 insecticidal protein DNA packaging into chromatin is necessary for stable chromosomal seg- which is known to be biologically active against several species within the Coleopteran regation, and also affects transcription. DNA is packaged around octa- family Chrysomelidae, including western corn rootworm, Diabrotica virgifera virgi- mers of histones, and histone and DNA modi®cations determine local fera LeConte, northern corn rootworm, D. barberi Smith and Lawrence, and Mexican chromatin states. Transitions among chromatin states occur via chromatin corn rootworm, D. v. zeae Krysan and Smith. YGRW expresses the Cry3Bb1 protein remodeling proteins. Protein motifs involved in chromatin modi®cation in the root system where larvae feed and in¯ict damage. Thus, the level of expression and remodeling are conserved across plants and animals. However, im- is critical to the control of the insects that come into contact with the root system. Through a series of experiments, it was shown that while the level of Cry3Bb protein portant differences are observed when comparing plant and animal pro- may vary between hybrids and over time, the level of protection is not affected by teins, and monocot versus dicot proteins. In this presentation, I will dis- such variation; that is, the expression level in commercial hybrids is suf®cient to cuss how our consortium has discovered putative chromatin proteins in provide consistent protection for CRW larval feeding. There are many bene®ts for plants using queries from diverse species. DNA methyltransferases, SET- growers, consumers and the environment associated with the commercialization of domain proteins, and methyl-binding domain proteins will be used to YGRW. YGRW hybrids are more ef®cacious than soil and seed-applied insecticides exemplify evolution of chromatin proteins. Functional consequences will in protecting roots from larval feeding damage. The Cry3Bb1 protein produced in the root does not require activation (as many conventional insecticides do), and its per- be discussed, and patterns of divergence will be highlighted. formance is unlikely to be impacted by severe environmental conditions. Varieties containing the Cry3BB1 protein are also combined through conventional breeding with other genetically enhanced maize varieties. Combinations with herbicide tolerance and lepidopteran insect protection provide hybrids to growers that offer more complete crop protection from the most economically damaging insect and weed pests. This performance and grower satisfaction has been demonstrated over the past 2 years of commercial experience. YGRW technology was initially launched in 2003 on approximately 400,000 acres. The number of acres have steadily increased over the past 2 years to approximately 4.5 to 5 million acres in 2005. Growers that have adopted YGRW technology have been extremely pleased with the performance and yield protection generated from planting these hybrids. In 2005 for example, most growers realized a signi®cant yield advantage over other control options. This advantage was likely augmented due to the severe drought in 2005 across much of the corn belt. The YGRW technology protected the roots from larval feeding allowing the roots to access water from a deeper zone within the soil pro®le.

In Vitro Biology Meeting 16-A Abstracts Plant Symposia

P-30 P-32 Allium Genomics: Exploiting Model Plants for Analyses of Enormous Plant Tissue Transformation Using Periodic Arrays of Vertically Aligned Nuclear Genomes. M. J. HAVEY. USDA-ARS and University of Wis- Carbon Nano®bers. T. E. MCKNIGHT, A. V. Melechko, G. D. Grif®n, consin, Madison, WI 53706. Email: [email protected] and M. L. Simpson. Oak Ridge National Laboratory, Oak Ridge, TN 37831. Email: [email protected] Enormous genomic resources have been developed for the grasses, cul- minating with the complete genomic sequence of rice and reduced-rep- Periodic arrays of vertically aligned carbon nano®bers (VACNFs) have resentation sequencing of maize. These extensive resources may be ap- been demonstrated as effective vectors for delivery of large molecules, plicable to other major groups of monocots outside of the grasses. The including DNA, into a variety of cell types. Using an approach that com- order Asparagales (carries the Alliums and asparagus) and the comme- bines the massive parallelism of whisker-mediated delivery and the pre- linids (carries the grasses) are sister monophyletic groups within the cision of microinjection, arrays of VACNF needles may be surface mod- monocots. The Alliaceae (onion, garlic, leek, chive, bunching onion, i®ed with material and simultaneously pressed into the intracellular do- among others) is the second most economically important family in the mains of large numbers of mammalian and plant cells. While a variety monocots, following only the Poaceae. The huge nuclear genomes of the of micromachined materials have been studied as gene delivery arrays Alliaceae are major constraints to the development of genomic resources. for both plant and animal cells, these materials often have been found to We sequenced asparagus and onion BACs and revealed high densities of have inadequate aspect ratio or lack mechanical strength to effectively retroelements and transposons with few open-reading frames. We also penetrate the plant cell wall. In contrast to these micromachined materials, observed little synteny on the recombinational and sequence levels among VACNFs feature a covalent bonding structure that provides strong, but asparagus, onion, and rice, as might be expected given that the Aspara- ¯exible, vertical elements well suited to the rigors of cellular interfacing. gales and commelinids split at least 130 million years ago. Nevertheless, They also have extremely high aspect ratio, with tip diameters of typically genomic resources developed for the grasses are useful for translational less than 100 nm and lengths up to many tens of microns. Mechanical genomics of the Alliums. Occasionally physically linked sequences in strength and high aspect ratio provides for effective penetration into cells, rice show genetic linkage in onion and this microsynteny across shorter including those protected by rugged cell walls such as yeast and pollen. genomic regions aids in the identi®cation and mapping of candidate The surface of nano®bers may be modi®ed with adsorbed or covalently genes. Single-copy expressed regions in the rice genome show signi®cant attached biomolecules and interfaced into cellular targets, including direct similarities and share most introns with coding regions in onion, allowing penetration into the nuclear domain. In this overview, we will describe the development of PCR-based markers carrying indels or single nucle- the fabrication and functionalization of VACNFs and the application of otide polymorphisms to evaluate for associations between candidate these modi®ed VACNFs as massively parallel delivery vectors for both genes and economically important traits. plant and animal cell transformation.

P-31 P-33 High Ef®ciency and High Throughput Transformation of Cereals Medi- DNA-coated Nanoparticles Mediated Transgene Expression in Plant ated by Agrobacterium for Functional Genomics. T. KOMARI. Plant In- Cells. FrancËois Torney1, Brian Trewyn2, Supratim Giri2, Victor Lin2 and novation Center, Japan Tobacco Inc., 700 Higashibara, Iwata, Shizuoka Kan Wang1. 1Center for Plant Transformation, Iowa State University, De- 438±0802, JAPAN. Email: [email protected] partment of Agronomy Ames, IA 50011 and 2Department of Chemistry, Iowa State University, Ames, IA 50011. Email: [email protected] Major cereals joined the list of plants that can be transformed by A. tumefaciens a decade ago. Then protocols for rice and maize have been Plant genetic engineering relies mostly on biolistic and Agrobacterium- tremendously improved. Overall ef®ciency has been increased at least 10 mediated transformation technologies. Both techniques allow DNA deliv- times, range of transformable genotypes widened, time for tissue culture ery into plant cells and subsequent integration into the genome. Recently, shortened, workload for tissue culture lessened, and related techniques the development of nanomaterials such as mesoporous silicate nanopar- developed like elimination of selectable markers from transformants and ticles (MSN) was shown to deliver marker genes into animal cells (Radu reduction of vector backbone transfer. The progress in transformation et al., 2004). The distinct feature of this nanoparticle is that it can both technology, which plays indispensable roles in applications including deliver DNA as well as chemicals encapsulated in the particles. Con- construction of T-DNA tagged lines, map-based cloning, characterization trolled release of the ®lling substance is also possible using this material of cloned genes and large-scale screening for gene effects, is a key factor (Gruenhagen et al., 2005). Here we show that this material can be used in current and future advances in cereal functional genomics. Throughput for transforming tobacco mesophyll protoplasts and immature maize em- of transformation in functional genomics must be very high. Rice is ideal bryos. Transgene expression was observed both transiently and stably. In in this context because of remarkable ef®ciency, short tissue culture pe- addition, chemicals encapsulated in the MSN can be controlled-released riods, and small workloads for tissue culture. Maize is also high in ef®- in planta when appropriate induction reagents are applied. The use of ciency of transformation but falls short of rice. Thus a possible option is mesoporous silicate nanoparticles to deliver transgenes and various sub- to screen gene effects initially in rice and to characterize selected genes stances simultaneously into plant cell opens a wide range of applications in maize. Taking advantage of the ef®ciency in rice, we have initiated a for future plant genomic study. high-throughput process to screen genomic fragments of plants that may Gruenhagen, J.A., Lai, C.Y., Radu, D.R., Lin, V.S., and Yeung, E.S. cause phenotypic changes. This is an approach in functional analysis of (2005). Real-time imaging of tunable adenosine 5-triphosphate release genomic sequences, which has recently been highlighted due to impor- from an MCM-41-type mesoporous silica nanosphere-based delivery sys- tance of non-protein-coding sequences and of multiple patterns of tran- tem. Appl Spectrosc 59, 424±431. scription in studies of eukaryotic genomes. This system was designated Radu, D.R., Lai, C.Y., Jeftinija, K., Rowe, E.W., Jeftinija, S., and Lin, as TraitExplorerTM because agronomically useful phenotypes identi®ed in V.S. (2004). A polyamidoamine dendrimer-capped mesoporous silica the process could be exploited as new traits in crop improvement. Initial nanosphere-based gene transfection reagent. J Am Chem Soc 126, 13216± characterization of transgenics has indicated that genomic fragments with 13217. unique functions may potentially be found by this method.

In Vitro Biology Meeting 17-A Abstracts Plant Symposia

P-34 P-36 Transfection: A Reliable and Ef®cient Method for Maize Transformation. Evaluation of an Automated Image Analysis System for Factors which M. E. HORN, J. A. Esser and G. Hall, Jr. Agrigenetics/Mycogen, 5649 Stabilize Gene Expression. J. M. CHIERA and J. J. Finer. Department of E. Buckeye Road, Madison, WI 53716. Email: michael.horn@ Horticulture and Crop Science, OARDC/The OhioState University, Woo- phytonbiotech.com ster, OH 44691. Email: [email protected] Maize has historically been amongst the most dif®cult of plant species A commonly observed phenomenon after transformation of plant tissues, to genetically engineer. Currently, particle bombardment (biolistics) and regardless of delivery method, is variable gene expression. This phenom- Agrobacterium tumefaciens are used to insert foreign genes into maize in enon requires the time consuming task of creating a large number of a stable manner. Both of these methods are covered by patents making clones in order to increase the probability of obtaining a successful trans- them expensive to practice for commercial purposes. We have developed formation event. The reasons for this variability in gene expression are a third method, one that is free of patent encumbrance, is reasonably unknown, but it is likely that protein or message instability, or transgene ef®cient, and produces regenerated transformed plants in a reasonable silencing is involved. With the advent of green ¯uorescent protein (GFP) mount of time. Callused immature zygotic embryos are incubated with and other ¯uorescent proteins, it is now possible to track transgene ex- naked DNA in a sterile cuvette for a given length of time and then elec- pression from the time of DNA introduction to plant recovery. The use troporated at a speci®ed voltage, capacitance, and pulse length and wave of ¯uorescent proteins in combination with automated image collection shape. A decay wave was successful whereas a square wave was not. and analysis software allows for the continuous monitoring and quanti- The protocol was optimized for transient expression of the GUS gene ®cation of in vivo gene expression in multiple tissue samples over time. and those parameters were used to produce stable transformants at a fre- With these tools, we can begin to dissect factors that positively or neg- quency of ϳ0.5%. The use of tobacco Rb7 matrix attachment regions atively affect transgene expression in transiently and stably transformed (MARs) gave a frequency of nearly 4% using the optimum transient tissues. We have evaluated sequences that appear to stabilize GFP ex- parameters. Further optimization of the electroporation parameters for pression in a cotyledon transient expression system. In control bombard- stable transformation resulted a transformation frequency of nearly 4% ments, without the additional sequences, GFP expression peaks 24 hours even without MARs. Further optimization could raise the transformation post bombardment and declines to minimal levels within 72 hours. Use frequencies even higher. Without MARs, single copy insertion events of GFP fused to some sequences led to extended GFP expression through composed about 50% of the total. With MARs, the proportion of single 168 hours. Co-introduction of these same sequences on different vectors copy insertion events was 90% of the total. This data implies that Trans- extended expression slightly but not to the same extent as the GFP-fu- fection is a viable transformation system for maize. sions. These factors may in¯uence gene expression by stabilizing protein, mRNA, or by suppression of host silencing.

P-35 P-37 A Novel Plant Transformation TechnologyÐLipoic Acid. YINGHUI Agrobacterium Mediated Gene Transfer in Plants. MARC VAN MON- DAN*. Monsanto Company, 700 Chester®eld Parkway, St. Louis, MO TAGU. Institute Plant Biotechnology for Developing Countries (IPBO), 63017 and *Current address: Institute for Advanced Learning and Re- Universiteit Gent, BELGIUM. Email: [email protected]; Website: search, and Departments of Horticulture and Forestry, Virginia Polytech- www.psb.UGent.be nic Institute and State University, 150 Slayton Avenue, Danville, VA The discovery that some Rhizobiaceae had evolved into pathogens ca- 24540. Email: [email protected] pable to genetically engineer a plant cell became an interesting chapter For the ®rst time a breakthrough in plant transformation technology has in the study of plant microbe interactions. The exploitation of this ca- been discovered in the antioxidant, lipoic acid (LA), found in most living pacity, now a good twenty years ago, to engineer novel traits into crop organisms. Utilizing LA in Agrobacterium-mediated transformation pro- plants, was a major breakthrough in fundamental and applied plant sci- cesses across ®ve different plant species has signi®cantly improved the ences. The possibility to add and later, thanks to the iRNA-technology, transformation methods, even for previously recalcitrant genotypes. Fre- to silence at will a set of genes in most plant species, got molecular plant quencies of soybean independent plant transgenic events were increased sciences off the ground. It also allowed the engineering of new traits in from 0.6 to 3.6%, potato from 3 to 19%, tomato from 28 to 94%, and some of the major crops. Now, global agriculture requires the extension wheat from 2.9 to 5.4%, and putative transgenic embryo frequency of of this technology to the improvement of barely domesticated crops. Un- cotton from 41 to 61%; frequency of escapes was reduced in soybean raveling the molecular base of plant growth and development, of stress from 92 to 72%, potato from 50 to 16% and tomato from 91 to 53% response and of biomass production is now becoming possible. Industry under the optimal conditions. This study also demonstrated that the in- already plans the production of new compounds and new materials in crease of the transformation frequency and reduction of escapes in tomato plants. To do the research and these applications successfully, we need a were accompanied by 2-fold reduction in severity of browning/necrosis substantial improvement of the ef®ciency of our universal gene vector of Agrobacterium-infected cotyledonary tissues, 2-fold increase in the Agrobacterium. We can ineed not rely on engineering of some model survivability of the transformed cotyledonary tissues, 4-fold increase in plants. We should be able to engineer the highest yielding cultivars and the percentage of transgenic shoots and 3-fold reduction of the percentage make that they can be grown in a sustainable way, this means with less of non-transgenic shoots when using LA under optimal conditions. LA water and nutrients than in today's agriculture. It is urgent that specialist application in plant transformation has dramatically resolved the three in ``Plant Cell and Tissue Culture'' take up this challenge. common problems in plant transformation: recalcitrance, tissue browning/ necrosis of the transformed cells/tissues, and escapes, which severely lim- it the number of transgenic plants that can be regenerated.

In Vitro Biology Meeting 18-A Abstracts